Device and method for determining the fluorescence and the number of antibodies on exosomes with mitigation of the intensity reduction of the fluorescent dye by laser light

Abstract

The invention relates to a device and method for reducing the reduction in intensity of a fluorescence dye by laser light when determining fluorescence and the number of antibodies on exosomes, comprising means for storing different measurement points of various differently coloured lasers in a measuring cell at certain measurement positions, the focussing of the laser beam interacting with the sample being recorded in a video camera as the centre of a convergent beam bundle.

Description

[0001] The application relates to a device for determining the fluorescence and number of antibodies on exosomes.

[0002] From DE 20 2018 005 287 U1 a device for determining the fluorescence and the number of antibodies on exosomes is known.

[0003] In biology, vesicles are intracellular, very small, round to oval sacs surrounded by a single or double membrane or a network-like protein shell. Vesicles form their own cellular compartments in which various cellular processes take place. They are approximately one micrometer in size. Vesicles are responsible for the transport of many substances within the cell.

[0004] The mechanisms leading to the formation of extracellular vesicles are not yet fully understood. Based on their origin or size, three types of extracellular vesicles are distinguished.

[0005] Exosomes are small vesicles with a size of approximately 50 to 150 nm.

[0006] According to claim 1 of the above-mentioned publication, the device has the following features: a) The beams from several different lasers (1, 2, 3, 4) are each directed separately by means of a separate collecting prism (14) onto a beam path (21) into a measuring cell (22) containing a sample (9) containing particles, wherein the focusing of the laser beam (21) in interaction with the sample (9) forms the center of a convergent beam bundle, consisting of light from the fluorescence plane (5) and the scattering plane (8), which, after passing through a liquid lens with an optical control (18), is registered in a video camera (15), b) the convergent beam path passes through a color filter (16) which is moved by means of a change wheel (17) and a control (26), c) a display (19) with a touchscreen (19) and an overall control (20) with a particle tracking program serve a video camera (15).

[0007] Multiple consecutive measurements with the same sample to obtain a statistically good result cannot be performed with the prior art described in DE 20 2018 005 287 U1, because the fluorescent dye used bleaches too much due to the long laser exposure time, necessitating a replacement of the sample in the measuring cell after each measurement. If the laser exposure time to the laser dye (fluorochrome) were shortened, the bleaching could be reduced, and multiple measurements with one and the same sample could be made possible.

[0008] The present application is therefore based on the task of shortening the exposure time of the laser light to the fluorochromes (fluorescent dye).

[0009] The problem is solved by the independent claims.

[0010] A device according to the invention comprises the features of claim 1.

[0011] Means are provided for storing different measurement points (26) of different different colored lasers in a measuring cell (17) at specific measurement positions, wherein the focusing of the respective laser beam (24) in interaction with the sample liquid (18) as the center of a convergent beam is registered in a video camera of the device.

[0012] A changer device (7), with a multi-notch filter (6) and a quartz glass 16, serves to adjust and secure the same optical path lengths for the fluorescence light and the laser scattered light in the convergent beam path between the liquid lens (15) and the measuring points 26 in the measuring cell (17).

[0013] A display (9) with a touchscreen and an overall control unit (10) with a particle tracking program serve to operate the video camera (13), wherein the video camera (13) preferably has a graphene-based light sensor (12) with an associated control unit (11) for the light sensor (12) and has fast stepper motors and backlash-free precision traversing carriages, and the camera is preferably a CMOS or an eCCD.

[0014] The method according to the invention is described in claim 4.

[0015] The measuring cell (17) is filled with test fluid to detect the different positions of the lasers used, their focusing and storage.

[0016] The calibration fluid is exchanged for sample fluid containing exosomes to be measured, and the absence of bubbles in the cell is checked (17). All lasers are then returned to their first position.

[0017] The lasers used are sequentially moved to their stored focus points and switched on at the start of the analysis, the images are captured by the light sensor (12) and the corresponding data are forwarded to the pattern recognition.

[0018] The figures in the application have the following content. Fig. 1: A representation of a specific NTA nanoparticle tracking method. Fig. 2: A representation of the measuring cell. Fig. 3: A representation of the decrease in dye intensity after laser exposure in seconds. Fig. 4: A representation of the measurement positions and the laser focus points (measurement points).

[0019] The Fig. 1 largely corresponds to the representation of Fig.1 from DE 20 2018 005 287 U1.

[0020] The number of lasers is five instead of four in the St.dT. The liquid filter 6 in the St.dT is replaced by a multi-notch filter 6 with an associated exchange device 7, for the multi-notch filter 6 and a quartz glass 16. The quartz glass 16 serves to adjust and ensure the same optical path lengths for the fluorescence light and the laser scattered light in the convergent beam path through the lens to the light sensor 12 during the adjustment of the measuring points 26 (see Figur 4 ) in measuring cell 17. The quartz glass simulates the same optical path lengths as the notch filter.

[0021] The video camera 15 at the St.dT has the number 13 here and features a graphene-based light sensor 12, with a control 11 for the light sensor 12, and controls a liquid lens 15 via the camera optics 14 and an optics control 8, which in turn hits a beam path 24 of one of the lasers 1 to 5 and one of the collecting prisms 21 via the notch filter 6 in the fluorescence plane 20 in the sample liquid 18 of the measuring cell 17 and the optics passage window 19 in the measuring cell 17.

[0022] The overall control 10 corresponds to the overall control 20 at the st.dT, where display 9 corresponds to the corresponding display 19.

[0023] The representation of measuring cell 17 in the Fig. 2 shows vertically from above, also in the Fig.1 The laser beam path shown, 24, which passes through the laser passage window 23, with the beam tapering to a point at the respective measuring point 26 visible to the side, which emerges from the measuring cell 17 through the quartz glass 16 and the liquid lens 5 and into the camera optics 14.

[0024] The filling opening 25 of the measuring cell 17 can be seen on the front.

[0025] The decrease in dye intensity after laser exposure in seconds is expressed as a percentage of the Fig. 3 The laser light should be applied to the fluorescence at measuring point 26 for a maximum of 2 seconds. After that, the intensity of the fluorescent dye becomes too weak and does not provide usable measurement results.

[0026] The representation of the measurement points 26 for the focus points (of the laser scattering plane and the fluorescence plane) of the optics for the beam path of the lasers 24 are shown in the various measurement positions 1 to 4. Fig. 4 The camera lens 14 is also shown here.

[0027] Examples include those in the Fig.4 The 4 positions shown are arranged according to the Fig. 1 carried out as follows.

[0028] Measuring cell 17 is filled with test fluid (also called calibration fluid), wherein this test fluid contains polystyrene particles or certified exosome standards instead of the exosome sample.

[0029] Im The individual lasers are then guided to the respective measuring point 26 of the measuring cell 17 (see Figur 4 ) and saved.

[0030] This means that all 5 lasers are each moved to measurement position 1, the optical path lengths are determined, the focus is applied, and then the data is saved. Then all 5 lasers are moved to the next measurement position, focused, and then the data is saved.

[0031] The same thing happens with position 3 and position 4 and all 5 lasers.

[0032] Then the lasers are switched off.

[0033] As a next step, the calibration fluid is replaced with a sample fluid containing exosomes in the measuring cell.

[0034] As a next step, the notch filter 6 is inserted between the liquid lens 15 and the measuring cell 17 and moved into the converging beam path, with the quartz glass 16 serving for fixation in conjunction with the device 7.

[0035] The actual measurement begins at measurement position 1 with the five lasers being switched on sequentially to their stored focus points, the images being captured by the light sensor, and the data being forwarded to the pattern recognition system. This takes less than two seconds. Afterwards, the lasers are switched off.

[0036] The lasers then move to measuring position 2. The measurement is carried out as in the previous step. Afterwards, the lasers are switched off.

[0037] The lasers move to measuring position 3. The measurement continues as in the previous step. Afterwards, the lasers are switched off.

[0038] The lasers move to measurement position 4. The data is then evaluated.

[0039] The particles can form different concentrations at various points within the measuring cell, for example, due to the filling process. Therefore, multiple measurement positions within the measuring cell are necessary to achieve good measurement quality and high statistical reliability.

[0040] In At the newly accessed measurement positions in the measuring cell, the fluorescences on the antibodies are still fresh and unused.

[0041] By switching off the lasers when moving to the next measurement position, the fluorescence is further protected, as no laser light can bleach the dyes. This is only possible by previously saving the individual measurement positions and focus points and by highly precise, rapid re-entry of the positions (fast stepper motors, backlash-free precision carriage for the lasers and the liquid lens in the objective).

[0042] The multibandpass filter 6 (multi-notch filter) enables the rapid switching of the individual lasers, resulting in a time saving for the preservation of the fluorescence, as no filter change is necessary.

[0043] The gentle measurement method shown allows for up to 100 or more measurement positions with measuring points 26 to be accessed and measured with five lasers on a single exosome fluorescence sample, not just four as in our example. This ensures very high measurement quality.

[0044] The camera must use a highly sensitive sCMOS or eCCD or graphene light sensor, as the multi-bandpass filter 6 (multi-notch filter) reduces the light intensity.

[0045] The method requires complex control of the described process and movement sequences by a special control and analysis program. Reference symbol list

[0046] 1 Laser 375 nm 2 Laser (violet = 405 nm) 3 Laser (blue = 488 nm) 4 Laser (green = 520 nm) 5 Laser (red = 640 nm) 6 Notch filter (multibandpass filter) 7 Exchange device for the notch filter and the quartz glass 8 Optics - control 9 Display with touchscreen 10 Overall control with particle tracking program 11 Control for light sensor 12 Graphene-based light sensor 13 Detector or video camera eCCD, sCMOS 14 Camera optics 15 Liquid lens with adjustable focus 16 Quartz glass 17 Measuring cell 18 Sample 19 Optics - transmission window of the measuring cell 17 20 Fluorescence plane 21 Converging prisms (guide all lasers into one beam path) 22 Scattered light plane 23 Laser - Through window of the measuring cell 17 24 Laser beam path 25 Filling opening of the measuring cell 17 26 Measuring point with the focal planes for laser scattered light and fluorescence light

Claims

1. A device for determining the fluorescence and the number of antibodies on exosomes, the device comprising: a measurement cell (17) for accommodating a sample liquid (18), a video camera (13) with camera optics (14), a liquid lens (15) with an adjustable focus, several varicolored lasers (1-5) and, for each laser (1-5), an associated collecting prism (21), wherein the device is configured to guide a beam from the respective laser (1-5) via the associated collecting prism (21) to a single common incident beam (24) for all the lasers (1-5), wherein the incident beam is incident into the measurement cell (17) at a measurement position 1, wherein the device is further configured to focus the incident beam (24) on a measurement point (26) within the measurement cell (17) and to register the focusing of the respective incident beam (24) in interaction with the sample liquid (18) in the measurement point (26) as a center of a convergent beam bundle after passing the liquid lens (15) in the video camera (13), a change device (7) with a filter (6) and a quartz glass (16) for setting and securing the same optical path lengths for fluorescence light and laser scattered light in a convergent beam path between the liquid lens (15) and the measurement point (26) in the measurement cell (17), a display (9) having a touchscreen and an overall controller (10) having a particle tracking program for operating the video camera (13), characterized in that the filter (6) is a multi-notch filter, and in that the device further comprises: means for moving to several predetermined measurement positions 1-4 with the lasers (1-5), so that the respective incident beam (24) is focusable on different measurement points (26) in the measurement cell (17); means for storing the different measurement positions 1-4 and associated focus points of the lasers (1-5); wherein the device is configured to move, with the lasers, to the stored measurement positions and the associated focus points of the lasers, and then to register the focusing of the respective incident beam (24) interacting with the sample liquid (18) in the respective measurement point (26) as center of a convergent beam bundle after passing through the liquid lens (15) in the video camera (13), and wherein the device is configured in such a manner that the lasers (1-5) are switched off when moving to the next measurement position 1-4.

2. The device according to claim 1, characterized in that the video camera (13) has a graphene-based light sensor (12) having an associated controller (11) for the light sensor (12).

3. The device according to any one of claims 1 or 2, characterized in that the device has rapid stepping motors and precision movement carriages without play, and the video camera (13) is an sCMOS or an eCCD.

4. A method for determining the fluorescence and the number of antibodies on exosomes by using a device according to any of the preceding claims, the method comprising the following steps: - filling the measurement cell (17) with a calibration liquid, - moving to and detecting the different measurement positions 1-4 of the lasers (1-5) that are used, focusing the lasers and storing the measurement positions 1-4 and the associated focus points of the lasers (1-5), wherein the measurement cell (17) is filled with the calibration liquid and the quartz glass (16) is positioned in the convergent beam path between the liquid lens (15) and the respective measurement point (26) within the measurement cell (17), - replacing the calibration liquid with a sample liquid having exosomes to be measured, and checking the measurement cell (17) for freedom from bubbles, - performing the following steps for all measurement positions 1-4 one after the other, wherein the measurement cell (17) is filled with the sample liquid and the multi-notch filter (6) is positioned in the convergent beam path between the liquid lens (15) and the respective measurement point (26) within the measurement cell (17), - moving to the respective measurement position 1-4 with the lasers (1-5), wherein the lasers that are used are moved successively to their stored focus points and are switched on at the beginning of an analysis, - registering the focusing of the respective laser beam (24) interacting with the sample liquid (18) in the measurement point (26) as center of a convergent beam bundle after passing through the liquid lens (15) in the video camera (13) with a light sensor (12) of the video camera (13), - forwarding corresponding data to a pattern recognition, wherein the respective lasers (1-5) are switched off when moving to the next measurement position.

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